Over the past several years it has become increasingly evident that cancer and normal development share many properties. Among other things, both processes involve alterations in cell proliferation and differentiation, alterations in cell death, neovascularization, cell motility, and invasion of surrounding tissue. Genes involved in these processes during normal development may therefore contribute to tumorigenesis if misexpressed. HSIX1 belongs to the superfamily of homeobox genes that encode transcription factors important for normal development, Its overexpression can attenuate the DNA damage-induced G2 cell cycle checkpoint in mammary carcinoma cells, providing evidence for its role in proliferative processes and suggesting a means through which it may affect tumorigenesis. Indeed, the gene is upregulated in 44 percent of primary breast cancers and 90 percent of metastatic lesions examined. Overexpression of HSIX1 in MCF7 cells significantly increases tumor burden in nude mice, suggesting that its role in cancer is causative, and not merely correlative. This proposal addresses the regulation of HSIX1, as well as the genes it regulates, in an effort to dissect the pathway (both upstream and downstream) through which HSIX1 affects cell cycle control and tumorigenesis. Specific aims 1 and 2 address the post-translational mechanisms by which the HSIX1 protein is regulated in the cell cycle in an effort to demonstrate whether this regulation is critical for its role in the G2 checkpoint and in tumorigenesis. Specifically, we will investigate how (a) mitotic phosphorylation and (b) proteasome-mediated degradation affect the role of HSIX1 in the G2 checkpoint and in tumorigenesis. This will be done using a variety of molecular biological, biochemical, and cell biological approaches, including irradiation assays in cell culture and nude mouse tumor assays. The last specific aim extends the proposal to identify genes transcriptionally regulated by HSIX1 in the G2 phase or at the G2/M boundary, with the goal of elucidating pathways important in HSIX1 control of the cell cycle and tumorigenesis. Methods will include examining known regulators of the G2 cell cycle checkpoint as well as microarray analysis. HSIX1 provides us with a unique opportunity to examine the relationship between developmental genes, cell cycle control, and cancer. A homeobox gene that is overexpressed in cancer cells but is normally absent or expressed at low levels in noncancerous, differentiated cells from the same tissue may serve as an ideal drug target, assuming that development of the organ is not essential at the time the cancer arises.